The membrane protein receptor is a protein that has a domain for penetrating the lipid double layer of a biological membrane to be present in a cell membrane, and specifically recognizes various physiological active substances to transmit and express their actions. The physiological active substance specifically binding to the membrane protein receptor is generally referred to as a ligand. The ligand is exemplified by a peptide hormone, a neurotransmitter, a growth factor and the like. The binding of the ligand to the membrane protein receptor causes a cell response via formation of second messenger, change in intracellular ion concentration, phosphorylation of proteins and the like. A series of reactions involving in changes such as formation of second messenger in cells, change in intracellular ion concentration and phosphorylation of proteins by binding of a ligand to the membrane protein receptor are generally referred to as a signal transduction, and a process for the series of reactions is referred to as a signal transduction pathway.
G-protein coupled receptor (hereunder, may be abbreviated as GPCR) is a glycoprotein that is present in cell membrane that is one kind of seven-span transmembrane receptor that has the structural characteristic of having seven cell membrane spanning domains, and it constitutes a super family with a many members. One thousand or more GPCR genes have already been identified, and studies are proceeding in relation to the three dimensional structure of GPCR, lignads for GPCR, intracellular signal transduction pathways through GPCR, and the functions thereof and the like.
GPCR is a receptor for light, odor and flavor and, at the same time, is also a hormone and neurotransmitter receptor and serves as an important sensor of cells in living organisms ranging from yeasts to humans.
When GPCR receives stimulation from a ligand it binds with G protein that is present inside the cell. G protein is a protein that couples with GPCR and has a function as a signal transduction factor. G protein is broadly classified into a several kinds of families based on functions to various factors (hereunder, referred to as “effector”) involved in signal transduction in the intracellular signal transduction pathways and difference in the genes encoding the protein. The G proteins that belong to each family are trimers comprising three subunits called α, β and γ, and normally guanosine 5′-diphosphate (GDP) is bound specifically to α-subunit. GDP-bound G protein is an inactive form that does not exhibit an action to an effector. When GPCR is stimulated by a ligand, an exchange reaction occurs between GDP binding to G protein and guanosine 5′-triphosphate (GTP) present in the cell, whereby the GDP is released from G protein and G protein then binds to GTP to form GTP-bound G protein. GTP-bound G protein is referred to as an active form, and it rapidly dissociates into α-subunit bound with GTP (αGTP) and a dimer (βγ) comprising β- and γ-subunits. αGTP and βγ directly act on an effector (for example, a calcium ion channel or a potassium ion channel) to activate the intracellular signal transduction pathway, and as a result, induce various cellular responses.
Amongst the GPCR superfamily, human brain angiogenesis inhibitor 2 (hereunder, abbreviated as hBAI2) is classified into class B (secretin like) and its gene is registered in GenBank under accession number AB 005298.
Although a report (Non-Patent Literature 1) exists relating to the sequence information and expression distribution of hBAI2, neither the function of hBAI2 nor its involvement in disease has been reported. However, based on a structural comparison with BAI1, a homolog of BAI2, it is considered that thrombospondin type I domain (hereunder referred to as “TSP-I domain”) is present in the extracellular domain (Non-Patent Literature 2). TSP-I domain is a characteristic domain recognized in a region comprising the amino acid sequence from position 385 to position 522 in thrombospondin, and it is known to be involved in the extracellular matrix of thrombospondin and as an important function domain for angiogenesis inhibiting ability.
Regarding hBAI1, it has been reported that its expression is specifically high in human brain tissue, that a domain having angiogenesis inhibitory ability is present in the extracellular region, that there is a possibility that it is subject to expression control by p53 and the like, suggesting the possibility that this gene is involved in some way in a mechanism relating to angiogenesis in the brain (Non-Patent Literature 1-4).
Regarding mouse BAI2 that is a gene associated with hBAI2, it is reported that a negative correlation is observed in the expression amount between BAI2 and vascular endothelial growth factor (VEGF) in brain tissue of cerebral ischemia model rat, and it is considered that, similarly to hBAI1, mouse BAI2 may be involved in angiogenesis. Specifically, expression of BAI2 was decreased after suffering the hypoxic state which was followed by increase of expression of VEGF. Further, a splicing variant of mouse BAI2 is reported to exist (non-patent document 3).
Even though it is predicted from the sequence information that both hBAI1 and hBAI2 belong to the GPCR family, no report can be found that mentions their functions as GPCR, including information regarding a ligand.
Meanwhile, cholecystokinin (hereunder, abbreviated as “CCK”) is known as a gastrointestinal hormone released from endocrine cell in duodenal mucous membrane. CCK is secreted accompanying intake of fat, and promotes gallbladder contraction and pancreatic enzyme secretion. It exhibits actions in the digestive organs including gallbladder contraction, promotion of pancreatic enzyme secretion and stimulation of intestinal movement. CCK is also considered a signaling substance that imparts a sensation of satiety to cerebral neurons.
CCK is known to be sulfated at the seventh tyrosine residue from the C-terminal side. By post-translational processing of CCK, fragments of several lengths that have different cleavage sites on the N-terminal side, such as CCK-4, CCK-8, CCK-12, CCK-33 and CCK-58, are produced. It has been verified that the physiological activity and amount of each of these fragments are different. Further, cerulein that is extracted from the skin of frog has been reported as a compound that has a similar chemical structure to CCK and which exhibits the same biological activity.
CCK is widely distributed in the brain and, for example, it has been observed in large amounts in brain cortex, hippocampus, amygdaloid body, and hypothalamus, and its action in the central nerves, such as involvement in anxiety, analgesia, sedation, food intake control, memory and learning is also reported. CCK is partially co-localized with DA (Dopamine) and GABA (γ-aminobutyric acid), and its interaction with 5HT (serotonin; 5-hydroxytryptamine)-functioned nervous system and the like has also been reported. It has also been reported that release of CCK is regulated by GABA. CCK-8 and CCK-4 have mainly been reported as CCK exhibiting bioactivity that is present in the brain. CCK-8 that is present in the brain is a cholecystokinin octapeptide sulfated form (CCK-8S) in which the seventh tyrosine residue from the C-terminal side is sulfated.
Recently, it has been reported that CCK is essential for memory retention. For example, it has been clarified that absence of CCK-8S makes it difficult to recall memory to conscious level and translate it into action, and that CCK-4 (a C-terminal tetrapeptide of CCK-33) obstructs mnemonic retrieval.
CCK-A receptor and CCK-B receptor have been reported as CCK receptors. These are both G-protein coupled receptors. Expression of CCK-A receptor is detected in tissues and cells originating in the alimentary canal, and in leukocytes and the like in the blood. CCK-A receptor is involved in alimentary regulation in the intracellular signal transduction pathway, for example, through promotion of effectors such as phospholipase C and adenyl cyclase. Meanwhile, expression of CCK-B receptor is detected in tissues and cells originating in the brain and alimentary canal, and in leukocytes and the like in the blood. CCK-B receptor is also referred to as “gastrin receptor”, and is involved in alimentary regulation and cell proliferation in the intracellular signal transduction pathway, for example, through promotion of effectors such as phospholipase C and intracellular calcium ion influx. In recent years, attention is being focused on the relation between CCK-B receptor and anxiety.
Non-Patent Literature 1: Shiratsuchi, T. et al., “Cytogenetics and cell genetics”, 1997, Vol. 79, p. 103-108.
Non-Patent Literature 2: Nishimori, H. et al., “Oncogene”, 1997, Vol. 15, p. 2145-2150.
Non-Patent Literature 3: Kee, H. J. et al., “Journal of Cerebral Blood Flow and Metabolism”, 2002, Vol. 22, p. 1054-1067.
Non-Patent Literature 4: Kaur, B. et al., “American Journal of Pathology”, 2003, Vol. 162, p. 19-27.